A method for the quantification of imipramine and its metabolites has been developed using HPLC. Utilizing this assay we studied the kinetics of imipramine metabolism in rats and in rat liver microsomes. The in vivo studies suggested that the conversion of desimipramine to hydroxy desimipramine after intramuscularly administered 3H-imipramine occurs more rapidly when oral unlabeled imipramine is also administered thatn when it is not. Studies with rat liver microsomes suggest that low doses of imipramine lead to an inactive cytochrome P-450 complex that is not formed when large doses of imipramine are administered.